Migraine is a common neurological disorder with high incidence in population. This study aimed to investigate the therapeutic efficacy of Tibetan medicine Ratanasampil(RNSP) and to identify the serum biomarkers for diagnosis and response assessment. We prospectively recruited 108 migraine patients living at high altitude (2,260 meters), including 40 patients for RNSP group, 40 patients for flunarizine(FLZ) group and 28 patients for placebo group. Serum levels of 5-hydroxytryptamine(5-HT), brain-derived neurotrophic factor(BDNF), Calcitonin gene related peptide(CGRP), nerve growth factor(NGF) and β-endorphin(β-EP) before and after therapy were measured. In comparison with placebo, both FLZ and RNSP significantly reduced the migraine days, HIT-6 score and verbal rating scale, headache intensity, duration, accompanying symptoms and headache score in four and eight weeks treatment. RNSP showed no significant difference to FLZ in the above parameters after four weeks treatment, but showed significantly better relief after eight weeks treatment. The overall effective rate of RNSP(92.5%) was also significantly higher than FLZ(74.4%,p < 0.05), mainly due to significantly higher ratio of patients with full recovery. The serum levels of biomarkers, including 5-HT,BDNF,NGF and β-EP, significantly elevated after eight weeks of treatment with RNSP, while the level of CGRP significantly decreased. The serum level of 5-HT exhibited significantly bigger percentage changes than other markers. In conclusion, RNSP was more effective than FLZ in relieving migraine after eight weeks continuous treatment. Serum 5-HT, BDNF, CGRP, NGF and β-EP were effective markers reflecting the response to RNSP and FLZ therapy.